Isolated nucleic acid molecules which encode GAGE genes and uses thereof

ABSTRACT

The invention relates to nucleic acid molecules which encode members of the GAGE family of tumor rejection antigen precursors and their use.

RELATED APPLICATION

This application is a divisional of Ser. No. 09/012,818, filed Jan. 23,1998, now abandoned, which is a continuation-in-part of Ser. No.08/531,662, filed Sep. 21, 1995, now U.S. Pat. No. 5,858,689, which is acontinuation-in-part of application Ser. No. 08/370,648, filed Jan. 10,1995, now U.S. Pat. No. 5,648,226, which is a continuation-in-part ofSer. No. 08/250,162 filed on May 27, 1994, now U.S. Pat. No. 5,610,013,which is a continuation-in-part of Ser. No. 08/096,039, filed Jul. 22,1993 now abandoned. All of these applications are incorporated byreference.

FIELD OF THE INVENTION

This invention relates to a nucleic acid molecule which codes for atumor rejection antigen precursor. More particularly, the inventionconcerns genes, whose tumor rejection antigen precursor is processed,inter alia, into at least one tumor rejection antigen that is presentedby HLA-Cw6 molecules. The genes in question do not appear to be relatedto other known tumor rejection antigen precursor coding sequences. Theinvention also relates to peptides presented by the HLA-Cw6 molecules,and uses thereof. Also a part of the inventions are peptides presentedby HLA-A29 molecules, and uses thereof.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T lymphocyte, or “T cell” response. This responserequires that T cells recognize and interact with complexes of cellsurface molecules, referred to as human leukocyte antigens (“HLAs”), ormajor histocompatibility complexes (“MHCs”), and peptides. The peptidesare derived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See in this regard Male et al.,Advanced Immunology (J. P. Lipincott Company, 1987), especially chapters6-10. The interaction of T cells and HLA/peptide complexes isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. This mechanism is involved in the immune system'sresponse to foreign materials, in autoimmune pathologies, and inresponses to cellular abnormalities. Much work has focused on themechanisms by which proteins are processed into the HLA bindingpeptides. See, in this regard, Barinaga, Science 257: 880 (1992);Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257:927 (1992); Latron et al., Science 257: 964 (1992). Also see Engelhard,Ann. Rev. Immunol. 12: 181-207 (1994).

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs cytolytic Tlymphocytes, or “CTLs” hereafter. The genes are said to code for “tumorrejection antigen precursors” or “TRAP” molecules, and the peptidesderived therefrom are referred to as “tumor rejection antigens” or“TRAs”. See Traversari et al., Immunogenetics 35: 145 (1992); van derBruggen et al., Science 254: 1643 (1991), for further information onthis family of genes. Also, see U.S. patent application Ser. No.807,043, filed Dec. 12, 1991, now U.S. Pat. No. 5,342,774.

In U.S. patent application Ser. No. 938,334, now U.S. Pat. No.5,405,940, the disclosure of which is incorporated by reference, it isexplained that the MAGE-1 gene codes for a tumor rejection antigenprecursor which is processed to nonapeptides which are presented by theHLA-A1 molecule. The reference teaches that given the known specificityof particular peptides for particular HLA molecules, one should expect aparticular peptide to bind to one HLA molecule, but not to others. Thisis important, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype. There is a need for further work in thearea, because cellular abnormalities are not restricted to oneparticular HLA phenotype, and targeted therapy requires some knowledgeof the phenotype of the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993, nowU.S. Pat. No. 5,558,995 and incorporated by reference, the fact that theMAGE-1 expression product is processed to a second TR is disclosed. Thissecond TRA is presented by HLA-C clone 10 molecules. The disclosureshows that a given TRAP can yield a plurality of TRAs.

U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, nowabandoned, and incorporated by reference herein teaches that tyrosinase,a molecule which is produced by some normal cells (e.g., melanocytes),is processed in tumor cells to yield peptides presented by HLA-A2molecules.

In U.S. patent application Ser. No. 08/032,978, now U.S. Pat. No.5,620,886, filed Mar. 18, 1993, and incorporated by reference in itsentirety, a second TRA, not derived from tyrosinase is taught to bepresented by HLA-A2 molecules. The TRA is derived from a TRAP, but iscoded for by a non-MAGE gene. This disclosure shows that a particularHLA molecule may present TRAs derived from different sources.

In U.S. patent application Ser. No. 08/079,110, now U.S. Pat. No.5,571,711, filed Jun. 17, 1993, and incorporated by reference herein, anunrelated tumor rejection antigen precursor, the so-called “BAGE”precursor, is described. The BAGE precursor is not related to the MAGEfamily.

The work which is presented by the papers, patents, and patentapplications cited supra deals, in large part, with the MAGE family ofgenes, and the unrelated BAGE gene. It has not been found, however, thatadditional tumor rejection antigen precursors are expressed by cells.These tumor rejection antigen precursors are referred to as “GAGE” tumorrejection antigen precursors. They do not show homology to either theMAGE family of genes or the BAGE gene. Thus the present inventionrelates to genes encoding such TRAPs, the tumor rejection antigenprecursors themselves as well as applications of both.

Thus, another feature of the invention are pepticies which are anywherefrom 9 to 16 amino acids long, and comprise the sequence:

Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa Tyr (SEQ ID NO:23)where Xaa is any amino acid. These peptides bind to, and/or areprocessed to peptides which bind to HLA-A29 molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D set forth lysis studies using CTL clone 76/6.

FIG. 2 shows tumor necrosis factor (“TNF”) release assays obtained withvarious transfectants and controls.

FIG. 3 compares lysis induced by cytolytic T lymphocytes of clone CTL76/6. Peptides of varying length were tested, including SEQ ID NOS: 4,5, 6, 7, 8 and 12.

FIGS. 4A-4B present an alignment of the cDNAs of the six GAGE genesdiscussed herein. In the figure, identical regions are surrounded byboxes. Translation initiation sites and stop codons are also indicated.Primers, used in polymerase chain reaction as described in the examplesare indicated by arrows.

FIG. 5 sets forth the alignment of deduced amino acid sequences for themember of the GAGE family. Identical regions are shown by boxes, and theantigenic peptide of SEQ ID NO: 4 is shown. SEQ ID NOS: 26, 27, 28, 29,30 and 31 are set out.

FIG. 6 shows the results obtained when each of the GAGE cDNAs wastransfected into COS cells, together with HLA-Cw6 cDNA. Twenty-fourhours later, samples of CTL 76/6 were added, and TNF release wasmeasured after twenty-four hours.

FIG. 7 compares the stimulation of CTL 22/23 by COS-7 cells, transfectedwith HLA-A29 cDNA, a MAGE, BAGE or GAGE sequence, as shown. Controlvalues are provided by MZ2-MEL.43 and COS cells, as stimulators.

FIG. 8 presents results obtained by ⁵¹Cr release studies, using variouspeptides including SEQ ID NO: 22 and various peptides derived therefrom.The peptides are amino acids 5-12, 4-12, 3-12, 6-13, 5-13, and 4-13 ofSEQ ID NO: 21.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

A melanoma cell line, MZ2-MEL was established from melanoma cells takenfrom patent MZ2, using standard methodologies. This cell line isdescribed, e.g., in PCT Application PCT/US92/04354, filed May 22, 1992,published Nov. 26, 1992, and incorporated by reference in its entirety.Once the cell line was established, a sample thereof was irradiated, soas to render it non-proliferative. These irradiated cells were then usedto isolate cytolytic T cell clones (“CTLs”) specific thereto.

A sample of peripheral blood mononuclear cells (“PBMCs”) was taken frompatent MZ2, and contacted to the irradiated melanoma cells. The mixturewas observed for lysis of the melanoma cells, which indicated that CTLsspecific for a complex of peptide and HLA molecule presented by themelanoma cells were present in the sample.

The lysis assay employed was a chromium release assay following Herin etat., Int. J. Cancer 39;390-396 (1987), the disclosure of which isincorporated by reference. The assay, however, is described herein. Thetarget melanoma cells were grown in vitro, and then resuspended at 10⁷cells/ml in DMEM, supplemented with 10 mM Hepes and 30% FCS, andincubated for 45 minutes at 37° C. with 200 μCi/ml of Na(⁵¹Cr)O₄.Labeled cells were washed three times with DMEM supplemented with 10 mMHepes. These were then resuspended in DMEM supplemented with 10 mM Hepesand 10% FCS, after which 100 ul aliquots containing 10³ cells, weredistributed into 96 well microplates. Samples of PBLs were added in 100ul of the same medium, and assays were earned out in duplicate. Plateswere centrifuged for 4 minutes at 100 g, and incubated for four hours at37° C. in an 8% CO₂ atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant werecollected and counted. Percentage of ⁵¹Cr release was calculated asfollows:${{\%\quad}^{51}{Cr}\quad{release}} = {\frac{\left( {{ER} - {SR}} \right)}{\left( {{MR} - {SR}} \right)} \times 100}$where ER is observed, experimental ⁵¹Cr release, SR is spontaneousrelease measured by incubating 10³ labeled cells in 200 ul of mediumalone, and MR is maximum release, obtained by adding 100 ul 0.3% TritonX-100 to target cells.

Those mononuclear blood samples which showed high CTL activity wereexpanded and cloned via limiting dilution, and were screened again,using the same methodology. The CTL clone MZ2-CTL 76/6 was thusisolated. The clone is referred to as “76/6” hereafter.

The same method was used to test target K562 cells, as well as themelanoma cell line. FIG. 1 shows that this CTL clone recognizes andlyses the melanoma cell line, i.e., MZ2-MEL but not K562. The clone wasthen tested against other melanoma cell lines and autologousEBV-transformed B cells in the same manner described supra. FIG. 1 showsthat autologous B cells, transformed by Epstein Barr Virus (“EBV”) werenot lysed, and that while MZ2-MEL 3.0 was lysed by CTL clone 76/6, thecell line MZ2-MEL.4F, a variant which does not express antigen F, wasnot. Hence, the clone appears to be specific for this antigen.

The results presented supra are inconclusive as to which HLA moleculepresents the TRA. The lysed cell line, i.e., MZ2-MEL, is known toexpress HLA-A1, HLA-A29, HLA-B37, HLA-B44, HLA-Cw6, and HLA-C clone 10.In experiments not reported here but which followed the protocol of thisexample, a subline of MZ2-MEL was tested, which had lost expression ofHLA molecules A29, B44, and C clone 10. The subline was lysed, thusindicating that the presenting molecule should be one of A1, B37 or Cw6.

EXAMPLE 2

Further studies were carried out to determine if 76/6 also producedtumor necrosis factor (“TNF”) when contacted with target cells. Themethod used was that described by Traversari et al., Immunogenetics 35:145-152 (1992), the disclosure of which is incorporated by reference.Briefly, samples of the CTL line were combined with samples of a targetcell of interest in culture medium. After 24 hours, supernatant from thecultures was removed, and then tested on TNF-sensitive WEHI cells. Cellline MZ2-MEL.43, a subclone of the MZ2-MEL cell line discussed supra aswell as in the cited references, gave an extremely strong response, andwas used in the following experiments.

EXAMPLE 3

The results from Example 2 indicated that MZ2-MEL.43 presented thetarget antigen of interest. As such, it was used as a source of totalmRNA to prepare a cDNA library.

Total RNA was isolated from the cell line. The mRNA was isolated usingan oligo-dT binding kit, following well recognized techniques. Once themRNA was secured, it was transcribed into cDNA, via reversetranscription, using an oligo dT primer containing a NotI site, followedby second strand synthesis. The cDNA was then ligated to a BstXIadaptor, digested with NotI, size fractionated by a Sephacryl S-500 HRcolumn, and then cloned, undirectionally, into the BstXI and NotI sitesof pcDNA I/Amp. The recombinant plasmid was then electroporated intoDH5α E. coli bacteria. A total of 1500 pools of 100 recombinant bacteriawere seeded in microwells. Each contained about 100 cDNAs, becausenearly all bacteria contained an insert.

Each pool was amplified to saturation and plasmid DNA was extracted byalkaline lysis and potassium acetate precipitation, without phenolextraction.

EXAMPLE 4

Following preparation of the library described in Example 3, the cDNAwas transfected into eukaryotic cells. The transfections, describedherein, were carried out in duplicate. Samples of COS-7 cells wereseeded, at 15,000 cells/well into tissue culture flat bottom microwells,in Dulbecco's modified Eagles Medium (“DMEM”) supplemented with 10%fetal calf serum. The cells were incubated overnight at 37° C., mediumwas removed and then replaced by 50 μl/well of DMEM medium containing10% Nu serum, 400 μg/ml DEAE-dextran, and 100 μM chloroquine, plus 100ng of the plasmids. As was indicated supra the lysis studies did notestablish which HLA molecule presented the antigen. As a result, cDNAfor each of the HLA molecules which could present the antigen (A1, B37,Cw6) was used, separately, to cotransfect the cells. Specifically, oneof 28 ng of the gene encoding HLA-A1 cloned into pCD-SRα, 50 ng of cDNAfor HLA-B37 in pcDNA 1/Amp, or 75 ng of cDNA for HLA-Cw6 in pcDNAI-Amp,using the same protocols as were used for transfeclion with the library.

Transfection was carried out in duplicate wells, but only 500 pools ofthe HLA-Cw6 transfectants could be tested in single wells. Followingfour hours of incubation at 37° C., the medium was removed, and replacedby 50 μl of PBS containing 10% DMSO. This medium was removed after twominutes and replaced by 200 μl of DMEM supplemented with 10% FCS.

Following this change in medium, COS cells were incubated for 24-48hours at 37° C. Medium was then discarded, and 1000-3000 cells of CTLclone 76/6 were added, in 100 μl of Iscove's medium containing 10%pooled human serum supplemented with 20-30 U/ml of recombinant IL-2.Supernatant was removed after 24 hours, and TNF content was determinedin an assay on WEHI cells, as described by Traversari et al.,Immunogenetics 35: 145-152 (1992), the disclosure of which isincorporated by reference.

The 1500 pools transfected with HLA-A1, and the 1500 pools transfectedwith HLA-B37 stimulated TNF release to a concentration of 15-20 pg/ml,or 2-6 pg/ml, respectively. Most of the HLA-Cw6 transfectants yielded3-20 pg/ml, except for one pool, which yielded more than 60 pg/ml. Thispool was selected for further work.

EXAMPLE 5

The bacteria of the selected pool were cloned, and 600 clones weretested. Plasmid DNA was extracted therefrom, transfected into a newsample of COS cells in the same manner as described supra, and the cellswere again tested for stimulation of CTL clone 76/6. Ninety-fourpositive clones were found. One of these, referred to as cDNA clone 2D6,was tested further. In a comparative test COS cells were transfectedwith cDNA clone 2D6 and the HLA-Cw6 cDNA, HLA-Cw6 cDNA alone, or cDNA2D6 alone. Control cell lines MZ2-MEL F and MZ2-MEL F⁺ were also used.TNF release into CTL supernatant was measured by testing it on WEHIcells, as referred to supra. The number of surviving WEHI cells wasmeasured by optical density after incubation of the cells with MTT. FIG.2 shows that the COS cells transfected with HLA-Cw6 and cDNA-2D6, andthe cell line MZ2-MEL F⁺ stimulated TNF release from CTL clone 76/6,indicating that HLA-Cw6 presented the subject TRA.

EXAMPLE 6

The cDNA 2D6 was sequenced following art known techniques. A sequencesearch revealed that the plasmid insert showed no homology to knowngenes or proteins. SEQ ID NO: 1 presents cDNA nucleotide information forthe identified gene, referred to hereafter as “GAGE”. A putative openreading frame is located at bases 51-467 of the molecule. The first twobases of this sequence are from the vector carrying the cDNA sequence,and are thus not part of the cDNA itself.

EXAMPLE 7

Following sequencing of the cDNA, as per Example 6, experiments werecarried out to determine it cells of normal tissues expressed the gene.To determine this, Northern blotting was carried out on tissues andtumor cell lines, as indicated below. The blotting experiments used cDNAfor the complete sequence of SEQ ID NO: 1, PCR was then used to confirmthe results.

TABLE 1 Expression of gene GAGE Normal tissues PHA activated T cells −CTL clone 82/30 − Liver − Muscle − Lung − Brain − Kidney − Placenta −Heart − Skin − Testis + Tumor cell lines Melanoma  7/16 Lung carcinoma1/6 Sarcoma 0/1 Thyroid medullary carcinoma 0/1 Tumor samples 1/1Melanoma

EXAMPLE 8

Detailed analysis of normal tissues and tumors was carried out byapplying polymerase chain reaction (“PCR”) and the GAGE gene informationdescribed supra.

First, total RNA was taken from the particular sample, using artrecognized techniques. This was used to prepare cDNA. The protocol usedto make the cDNA involved combining 4 ul of reverse transcriptase buffer5×, 1 ul of each dNTP, (10 mM), 2 ul of dithiothreitol (100 mM), 2 ul ofdT-15 primer (20 um), 0.5 ul of RNasin (40 units/ul), and 1 ul of MoMLVreverse transcriptase (200 units/ul). Next, 6.5 ul of template RNA (1ug/3.25 ul water, or 2 ug total template RNA) was added. The totalvolume of the mixture was 20 ul. This was mixed and incubated at 42° C.for 60 minutes, after which it was chilled on ice. A total of 80 ul ofwater was then added, to 100 ul total. This mixture was stored at −20°C. until used in PCR.

To carry out PCR, the primers

-   -   5′-AGA CGC TAC GTA GAG CCT-3′        (sense) and    -   5′-CCA TCA GGA CCA TCT TCA-3′        (antisense)        SEQ ID NOS: 2 and 3, respectively, were used. The reagents        included 30.5 ul water, 5 ul of PCR buffer 10×, 1 ul of each        dNTP (10 uM), 2.5 ul of each primer (20 uM), and 0.5 ul of        polymerizing enzyme Dynazyme (2 units/ul). The total volume was        45 ul. A total of 5 ul of cDNA was added (this corresponded to        100 ng total RNA). The mixture was combined, and layered with        one drop of mineral oil. The mixture was transferred to a        thermocycler block, preheated to 94° C., and amplification was        carried out for 30 cycles, each cycle consisting of the        following:

first denaturation: 94° C., 4 min. denaturation: 94° C., 1 min.annealing: 55° C., 2 min. extension: 72° C., 3 min. final extension: 72°C., 15 min.Following the cycling, 10 ul aliquots were run on a 1.5% agarose gel,stained with ethidium bromide.

cDNA amplified using the primers set forth supra yields a 238 base pairfragment. There is no amplification of contaminating genomic DNA, ifpresent.

The results are presented in Table 2, which follows. They confirm thatthe only normal tissue which expresses GAGE is testis, whereas a numberof tumors, including melanoma, lung, breast, larynx, pharynx, sarcoma,testicular seminoma, bladder and colon express the gene. Thus, any oneof these tumors can be assayed for expression of the GAGE gene.

TABLE 2 RT-PCR Analysis of the Expression of Gene GAGE NORMAL TISSUESHeart − Brain − Liver − Lung − Kidney − Spleen − Lymphocytes − Bonemarrow − Skin − Naevus − Melanocytes − Fibroblasts − Prostate − Testis +Ovary − Breast − Adrenals − Muscle − Placenta − Umbilical cord − TUMORSCell Lines Tumor Samples Melanoma 40/63 46/146 (32%) Lung cancerEpidermoid carcinoma 10/41 (24%) Adenocarcinoma 4/18 Small Cell LungCancer  6/23 0/2 Breast cancer 15/146 (10%) Head and neck tumor Larynx6/15 (40%) Pharynx 3/13 Sarcoma 1/4 6/18 (33%) Testicular seminoma 6/6(100%) Bladder cancer 5/37 (14%) Prostate cancer 2/20 Colon cancer  5/130/38 Renal cancer 0/6 0/45 Leukemia 3/6 0/19

EXAMPLE 9

The identification of the nucleic acid molecule referred to in the priorexamples led to further work directed to determination of tumorrejection antigens presented by HLA-Cw6 molecules, and derived from theGAGE gene.

The complete cDNA of GAGE in expression vector pcDNA/AmpI was digestedwith restriction endonucleases NotI and SpHI, and then with exonucleuaseIII following supplier's instruction (Erase-a-base System, Promega).This treatment generated a series of progressive deletions, staring atthe 3′ end.

The deletion products were ligated back into pcDNA/AmpI, and thenelectroporated into E.coli strain DH5α′IQ, using well known techniques.The transformants were selected with ampicillin (50 micrograms/ml).

Plasmid DNA was extracted from each recombinant clone and was thentransfected into COS-7 cells, together with a vector which coded forHLA-Cw6. The protocol used followed the protocols described above.

The transfectants were then tested in the TNF release assay. Thispermitted separation of positive and negative clones. All the negativeclones showed a deletion of the entire GAGE sequence. The smallestpositive clone contained the first 170 nucleotides of SEQ ID NO: 1. Theanalysis of this sequence, supra, notes that the open reading framestarts at nucleotide 51. Thus, this fragment contains a sequence whichencodes the first 40 amino acids of the GAGE TRAP.

EXAMPLE 10

Additional experiments were then carried out to define the regionencoding the TRA peptide more precisely. Polymerase chain reaction(“PCR”) amplification was used to do this.

Two primers were synthesized. The first primer was a 22-mercomplementary to a sequence within the plasmid vector pcDNAI/Amp locatedupstream of a BamHI site. The second primer was a 29-mer containing atthe 3′ end nucleotides 102-119 of SEQ ID NO: 1, and at the 5′ end anextension of 11 nucleotides containing an XbaI restriction site.

Following amplification, the PCR product was digested by BamHI and XbaI,and cloned into the BamHI-Xbal sites of plasmid pcDNA-3. The recombinantcolonies were cotransfected into COS-7 cells with cDNA encoding HLA-Cw6,in accordance with Example 4, and a TNF release assay, also as describedsupra, was carried out, using CTL 76/6.

TNF release was observed, indicating that the “minigene” was processedto a TRA. The minigene, i.e., nucleotides 1-119 of SEQ ID NO: 1, thecoding region of which runs from nucleotides 51-119, encoded the first23 amino acids of the cDNA of SEQ ID NO: 1. This information served asthe basis for the next set of experiments.

EXAMPLE 11

Two peptides were synthesized, based upon the first 23 amino acids ofSEQ ID NO: 1. These were:

Met Ser Trp Arg Gly Arg Ser Thr Tyr Arg Pro Arg Pro Arg Arg (SEQ IDNO:12) and Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val Glu Pro Pro Glu MetIle (SEQ ID NO:13)Each peptide was pulsed into COS-7 cells previously transfected withHLA-Cw6 cDNA, and combined with CTL 76/6 to determine if TNF releasewould be induced. Peptides (20 ug/ml) were added to COS-7 cells whichhad been transfected with the HLA-Cw6 cDNA twenty-four hours previouslyAfter incubation at 37° C. for 90 minutes, medium was discarded, and3000 CTLs were added in 100 microliters of medium, containing 25units/ml of IL-2. Eighteen hours later, TNF content of supernatant wastested via determining toxicity on WEHI-164-13 cells. The second peptide(SEQ ID NO: 13) was found to induce more than 30 pg/ml of TNF, while thefirst peptide (SEQ ID NO: 12) was found to induce less than 10 pg/ml ofTNF. The second peptide was used for further experiments.

EXAMPLE 12

Various peptides based upon SEQ ID NO: 13 were synthesized, and tested,some of which are presented below. To carry out these tests ⁵¹Crlabelled LB33-EBV cells, which are HLA-Cw6 positive, were incubated withone of the following peptides:

Tyr Arg Pro Arg Pro Arg Arg Tyr (SEQ ID NO:4) Thr Tyr Arg Pro Arg ProArg Arg Tyr (SEQ ID NO:5) Thr Arg Pro Arg Pro Arg Arg Tyr Val (SEQ IDNO:6) Thr Tyr Arg Pro Arg Pro Arg Arg Tyr Val (SEQ ID NO:7) Arg Pro ArgPro Arg Arg Tyr Val Glu (SEQ ID NO:8) Met Ser Trp Arg Gly Arg Ser ThrTyr Arg Pro Arg Pro Arg Arg (SEQ ID NO:12)The peptide concentration varied, as indicated in FIG. 3, and the ratioof CTL: LB33-EBV (“effector:target ratio”), was 10:1. ⁵¹Cr release wasdetermined after four hours of incubation at 37° C. Levels of lysis forpositive (“F⁺”, MZ2-MEL.3.1), and negative (“F”; MZ2-MEL.2.2.5) controlcells are indicated, in FIG. 3.

It was found, quite surprisingly that the octamer of SEQ ID NO: 4 wasthe best peptide, and appeared to be the tumor rejection antigen. Thisis the first time an octamer has been reported as being involved inpresentation by a human MHC molecule. There is some precedent for amurine system, as reported by Engelhard, supra, at 199, for H-2K^(b) andH-2K^(K) molecules. The nonamers of SEQ ID NO: 5 and SEQ ID NO: 6 alsoinduced CTL lysis albeit to a lesser extent than the octamer of SEQ IDNO: 4.

In results not reported here, a second CTL was tested (CTL 82/31). ThisCTL was known to lyse cells presenting MZ2-F. It, too, lysed HLA-Cw6positive cells following pulsing with the peptide of SEQ ID NO: 4.

EXAMPLE 13

To find out whether the GAGE DNA set forth supra was unique, a cDNAlibrary made with RNA from MZ2-MEL.43 (the same library that was usedfor the cloning of GAGE) was hybridized with a probe derived from theGAGE cDNA. The probe was a PCR fragment of 308 base pairs betweenpositions 20 and 328 of SEQ ID NO: 1. Twenty positive cDNAs wereobtained. Six of them were entirely sequenced. They were all highlyrelated to the GAGE sequence, but they were slightly different from it.Two of the six clones were identical to each other, but all the othersdiffered from each other. Thus, five new sequences different from buthighly related to GAGE were identified. They are called GAGE-2, 3, 4, 5and 6 (FIG. 4) and are presented as SEQ ID NOS: 14-18, 18, respectively.The fourteen other clones were partially sequenced at the 5′ end andtheir sequence corresponded to one of the six GAGE cDNAs.

The major difference between these cDNAs and GAGE-1 is the absence of astretch of 143 bases located at position 379 to 521 of the GAGE sequenceof SEQ ID NO: 1. The rest of the sequences shows mismatches only at 19different positions, with the exception of GAGE-3 whose 5′ end istotally different from the other GAGE for the first 112 bases. Thisregion of the GAGE-3 cDNA contains a long repeat and a hairpinstructure.

The deduced GAGE-1 protein corresponding to a tumor rejection antigenprecursor is about 20 amino acids longer than the 5 other proteins,whose last seven residues also differ from the homologous residues ofGAGE-1 (FIG. 5). The rest of the protein sequences show only 10mismatches. One of these is in the region corresponding to the antigenicpeptide of SEQ ID NO: 4. The sequence of the peptide is modified inGAGE-3, 4, 5 and 6 so that position 2 is now W instead of R.

EXAMPLE 14

To assess whether the change at position 2 affected the antigenicity ofthe peptide, cDNA of the 6 GAGE cDNAs were individually transfected intoCOS cells together with the cDNA of HLA-Cw6, and the transfectants weretested for recognition by CTL 76/6 as described, supra. Only GAGE-1 andGAGE-2 transfected cells were recognized, showing that the modifiedpeptide encoded by GAGE-3, 4, 5 and 6 was not antigenic in the contextof this experiment. Sequence analysis of the 5′ end of the 14 otherclones mentioned supra showed that 7 of them contained the sequenceencoding the antigenic peptide, and thus probably corresponded to eitherGAGE-1 or GAGE-2.

EXAMPLE 15

The PCR primers used supra to test the expression of GAGE in tumorsamples do not discriminate between GAGE-1 or 2 and the four other GAGEcDNAs that do not encode antigen MZ2F. A new set of primers was preparedwhich specifically amplifies GAGE-1 and 2, and not GAGE-3, 4, 5 and 6.These primers are:

VDE44 5′-GAC CAA GAC GCT ACG TAG-3′ (SEQ ID NO:9) VDE24 5′-CCA TCA GGACCA TCT TCA-3′ (SEQ ID NO:10)These primers were used as described, supra, in a RT-PCR reaction usinga polymerase enzyme in the following temperature conditions:

40 min at 94° C. 30 cycles with 1 min at 94° C. 2 min at 56° C. 3 min at72° C. 15 min at 72° C.The results of this analysis are set forth in Table 3.

TABLE 3 Expression of GAGE genes by tumor samples and tumor cell linesNumber of GAGE positive tumors Histological type All GAGE genes* GAGE-1and 2** Tumor samples Melanomas primary lesions 5/39  5/39 (13%)metastases 47/132 36/131 (27%) Sarcomas 6/20  6/20 (30%) Lung carcinomaNSCLC 14/65  12/64 (19%) Head and neck squamous 13/55  10/54 (19%) cellcarcinomas Prostatic carcinomas 2/20  2/20 Mammary carcinomas 18/16214/162 (9%) Bladder carcinomas superficial 1/20  1/20 infiltrating 5/26 3/26 Testicular seminomas 6/6   5/6 Colorectal carcinomas 0/43Leukemias and lymphonas 0/25 Renal carcinomas 0/46 Tumor cell linesMelanomas 45/74  40/74 (54%) Sarcomas 1/4   1/4 Lung carcinomas SCLC7/24  7/24 (29%) NSCLC 1/2   1/2 Mesotheliomas 5/19  5/19 (26%) Head andneck squamous 0/2  cell carcinomas Mammary carcinomas 1/4   0/4 Bladdercarcinomas 0/3  Colon carcinomas 5/13  5/13 Leukemias 3/6   1/6Lymphomas 0/6  Renal carcinomas 0/6  *Expression of GAGE was tested byRT-PCR on total RNA with primers VDE-18 and VDE-24, detecting all GAGEgenes. No PCR product was observed when these primers were assayed onDNA from MZ2-MEL. **Expression of GAGE-1 and 2 was tested by RT-PCR ontotal RNA with primers VDE-44 and VDE-24, which distinguish GAGE-1 and 2from the four other GAGE genes. No PCR product was observed when theseprimers were assayed on DNA from MZ2-MEL.

In further work, new primers were designed which amplified all GAGEgenes, to make sure that there was no expression of any of them innormal tissues. These primers are

VDE43 5′-GCG GCC CGA GCA GTT CA-3′ (SEQ ID NO:11) VDE24 5′-CCA TCA GGACCA TCT TCA-3 (SEQ ID NO:10)These were used exactly as for the PCR using the VDE44 and VDE24primers. The results are shown in Table 4. They confirm that the normaltissues are negative, except for testis.

TABLE 4 Expression of GAGE genes in normal adult and fetal tissues GAGEexpression* Adult tissues Adrenal gland − Benign naevus − Bone marrow −Brain − Breast − Cerebellum − Colon − Heart − Kidney − Liver − Lung −Melanocytes − Muscle − Ovary − Prostate − Skin − Splenocytes − Stomach −Testis + Thymocytes − Urinal bladder − Uterus − Placenta − Umbilicalcord − Fetal tissues* Fibroblasts − Brain − Liver − Spleen − Thymus −Testis + *Expression of GAGE was tested by RT-PCR amplification on totalRNA with primers VDE43 and VDE24 detecting all GAGE genes (FIG. 7).Absence of PCR product is indicated by − and presence by +. No PCRproduct was observed when these primers were assayed on DNA fromMZ2-MEL. *Fetal tissues derive from fetuses older than 20 weeks.

EXAMPLE 16

In work not reported here, it had been ascertained that cytolytic T cellclone CTL 22/23 (Van den Eynde, et al., Int. J. Cancer 44: 634-640(1989), incorporated by reference) did not recognize melanoma cellMZ2-MEL.3.1. This melanoma cell line was reported by Van der Bruggen, etal., Eur. J. Immunol. 24: 2134-2140 (1994), to have lost expression ofMHC molecules HLA-A29, HLA-B24, and HLA-Cw*1601. Studies were undertakento determine if transfection with one of these MHC molecules couldrender the line sensitive to CTL 22/23. HLA-A29 was the first moleculetested. To do so, poly A⁺ RNA was extracted from HLA-A29⁺ cell lineMZ2-MEL.43, using a commercially available extraction kit, and followingthe manufacturer's instructions. The mRNA was then converted to cDNA,using standard methodologies, size fractionated, and then insertedunidirectionally, into the BstXI and NotI sites of plasmid pcDNA-I/Amp.The plasmids were electroplated into E. coli strain DH5α5′IQ, andselected with ampicillin (50 μg/ml). The bacteria were plated ontonitrocellulose filters, and duplicated. The filters were prepared, andhybridized overnight in 6×SSC/0.1% SDS/1×Denhardt's solution at 40° C.,using ³²P labelled probe:

5′-ACTCCATGAGGTATTTC-3′ (SEQ ID NO:19)The probe is a sequence which surrounds the start codon of most HLAsequences.

The filters were washed twice, at room temperature for 5 minutes eachtime in 6×SSC, and twice in 6×SSC at 43° C. Positive sequences were thenscreened with probe:

5′-TTTCACCACATCCGTGT-3′ (SEQ ID NO:20)which had been labelled with ³²P. This sequence is specific for HLA-A29,as determined by reference to the Kabat Database of sequences andproteins of immunological interest, incorporated by reference.

This database is available at the NCBI (USA), or on Web Site (Internet)WWW.NCBI.NLM.NIH.GOV. The filters were washed twice at room temperaturefor 5 minutes each time, at 6×SSC, followed by two washes, at 6×SSC (5minutes per wash), at 42° C.

EXAMPLE 17

Once positive HLA-A29 clones were isolated, these were transfected intoCOS-7 using the DEAE-dextran chloroquine method out supra. In brief,1.5×10⁴ COS-7 cells were treated with 50 ng of plasmid pcDNA-I/Ampcontaining HLA-A29, and 100 ng of cDNA containing cDNA for one of theGAGE sequences mentioned supra, or one of the prior art MAGE or BAGEsequences in plasmid pcDNAα-I/Amp or pcDSR-α, respectively. Thetransfectants were then incubated for 24 hours at 37° C.

The transfectants were then tested for their ability to stimulate TNFproduction by CTLs, using the assay explained at the end of example, 4,supra.

FIG. 7, which presents the results of this study, shows that high levelsof TNF production were achieved using any of GAGE-3, 4, 5 or 6 andHLA-A29 as transfectants. GAGE-1 and GAGE-2, in contrast, do notstimulate CTL clone 22/23, thus leading to the conclusion that GAGE 3,4, 5 and 6 are processed to an antigen or antigens presented by HLA-A29molecules and recognized by CTL 22/23.

EXAMPLE 18

The fact that GAGE-3, 4, 5 and 6 were processed to peptides presented byHLA-A29+ cells, while GAGE-1 and GAGE-2 were not, suggested examinationof the deduced amino acid sequences for those common to GAGE 3, 4, 5 and6 and absent from GAGE-1 and GAGE-2. The sequence:

Arg Ser Thr Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr Val Gln (SEQ ID NO:21)was identified. The peptide was synthesized, lyophilized, and thendissolved in 1 volume DMSO, 9 volumes of 10 mM acetic acid in water.This methodology was used for the other peptides synthesized, discussedinfra.

The peptide (SEQ ID NO: 21) was tested in a ⁵¹Cr release experiment,following the method described supra.

It was found that this peptide did provoke lysis. Successive deletionswere prepared, and tested for their ability to provoke lysis, againusing the ⁵¹Cr lytic assay. This work is depicted in FIG. 8. It wasfound that the shortest peptide to provoke lysis was

-   -   Tyr Tyr Trp Pro Arg Pro Arg Arg Tyr        (SEQ ID NO: 22), which is common to all of GAGE-3 through 6.        Specifically, amino acids 10-18 of GAGE-3, and amino acids 9-17        of GAGE-4, 5 and 6 correspond to this peptide.

The members of the peptide family shown in FIG. 8, and represented,e.g., by SEQ ID NOS: 21 and 22, do not accord with the data presented byToubert, et al., “HLA-A29 Peptide Binding Motif”, Abstract No. 4183,Ninth International Congress of Immunology, Jul. 23-29, 1995, SanFrancisco, Calif., incorporated by reference. According to Toubert, etal., at the least a Phe residue is required at the third position of anypeptide which binds to HLA-A29. As is shown herein, such is not thecase.

The foregoing examples show the isolation of nucleic acid moleculeswhich code for tumor rejection antigen precursors and tumor rejectionantigens. These molecules, however, are not homologous with any of thepreviously disclosed MAGE and BAGE coding sequences described in thereferences set forth supra. Hence, one aspect of the invention is anisolated nucleic acid molecule which comprises the nucleotide sequencesset forth in any of SEQ ID NOS: 1-6 as well as fragments thereof, suchas nucleotides 1-170, and 51-170 of SEQ ID NO: 1, or any other fragmentwhich is processed to a tumor rejection antigen. The sequences of SEQ IDNOS: 1-6 are neither MAGE nor BAGE coding sequences, as will be seen bycomparing those to the sequence of any of these genes as described inthe cited references. Also a part of the invention are those nucleicacid molecules which also code for a non-MAGE and non-BAGE tumorrejection antigen precursor but which hybridize to a nucleic acidmolecule containing the described nucleotide sequence of SEQ ID NO: 1,under stringent conditions. The term “stringent conditions” as usedherein refers to parameters with which the art is familiar. Morespecifically, stringent conditions, as used herein, refers tohybridization in 1M NaCl, 1% SDS, and 10% dextran sulfate for 18 hoursat 65° C. This is followed by two washes of the filter at roomtemperature for 5 minutes, in 2×SSC, and one wash for 30 minutes in2×SSC, 0.1% SDS, at 65° C. There are other conditions, reagents, and soforth which can be used, which result in the same or higher degree ofstringency. The skilled artisan will be familiar with such conditionsand, thus, they are not given here.

It will also be seen from the examples that the invention embraces theuse of the sequences in expression vectors, as well as to transform ortransfect host cells and cell lines, be these prokaryotic (e.g., E.coli), or eukaryotic (e.g., CHO or COS cells). The expression vectorsrequire that the pertinent sequence, i.e., those described supra, beoperably linked to a promoter. As it has been found that both of humanleukocyte antigens HLA-Cw6 and HLA-A29 present tumor rejection antigensderived from these genes, the expression vector may also include anucleic acid molecule coding for one of HLA-Cw6 or HLA-A29. In asituation where the vector contains both coding sequences, it can beused to transfect a cell which does not normally express either one. Thetumor rejection antigen precursor coding sequence may be used alone,when, e.g., the host cell already expresses one or both of HLA-Cw6 andHLA-A29. Of course, there is no limit on the particular host cell whichcan be used. As the vectors which contain the two coding sequences maybe used in HLA-A29 or HLA-Cw6 presenting cells if desired, and the genefor tumor rejection antigen precursor can be used in host cells which donot express HLA-A29 or HLA-Cw6.

The invention also embraces so called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

To distinguish the nucleic acid molecules and the TRAPs of the inventionfrom the previously described MAGE and BAGE materials, the inventionshall be referred to as the GAGE family of genes and TRAPs. Hence,whenever “GAGE” is used herein, it refers to the tumor rejection antigenprecursors coded for by the previously described sequences. “GAGE codingmolecule” and similar terms are used to describe the nucleic acidmolecules themselves.

The invention as described herein has a number of uses, some of whichare described herein. First, the invention permits the artisan todiagnose a disorder such as melanoma, characterized by expression of theTRAP, or presentation of the tumor rejection antigen. These methodsinvolve determining expression of the TRAP gene, and/or TRAs derivedtherefrom, such as a TRA presented by HLA-Cw6 or HLA-A29. In the formersituation, such determinations can be carried out via any standardnucleic acid determination assay, including the polymerase chainreaction, or assaying with labelled hybridization probes. In the lattersituation, assaying with binding partners for complexes of TRA and HLA,such as antibodies, is especially preferred. An alternate method fordetermination is a TNF release assay, of the type described supra. Tocarry out the assay, it is preferred to make sure that testis cells arenot present, as these normally express GAGE. This is not essential,however, as one can routinely differentiate between testis and othercell types. Also, it is practically impossible to have testis cellspresent in non-testicular sample.

The isolation of the TRAP gene also makes it possible to isolate theTRAP molecule itself, especially TRAP molecules containing the aminoacid sequence coded for by any of SEQ ID NOs: 2-6. These isolatedmolecules when presented as the TRA, or as complexes of TRA and HLA,such as HLA-Cw6 or HLA-A29, may be combined with materials such asadjuvants to produce vaccines useful in treating disorders characterizedby expression of the TRAP molecule.

Exemplary adjuvants include Freund's complete and incomplete adjuvant,killed B. pertussis organisms, “BCG”, or Bacille Calmente-Guerin,Al(OH)₃, muramyl dipeptide and its derivatives, which may be emulsifiedin metabolizable oils, such as squalene, monophosphoryl lipid A (MPL),keyhole limpet hemocyanin (KLH), saponin extracts such as QA-7, QA-19,and QA-21 (also referred to as QS-21), these having been described inU.S. Pat. No. 5,057,540 to Kensil, et al., incorporated by reference,MTP-MF59, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), the cationic amphiphile DOTMA, the neutralphospholipids such as DOPE, and combinations of these. This listing isby no means comprehensive, and the artisan of ordinary skill will beable to augment this listing. All additional adjuvants are encompassedherein.

In addition, vaccines can be prepared from cells which present theTRA/HLA complexes on their surface, such as non-proliferative cancercells, non-proliferative transfectants, et cetera. In all cases wherecells are used as a vaccine, these can be cells transfected with codingsequences for one or both of the components necessary to provide a CTLresponse, or be cells which express both molecules without transfection.Further, the TRAP molecule, its associated TRAs, as well as complexes ofTRA and HLA, may be used to produce antibodies, using standardtechniques well known to the art.

When “disorder” is used herein, it refers to any pathological conditionwhere the tumor rejection antigen precursor is expressed. An example ofsuch a disorder is cancer, melanoma in particular. Melanoma is wellknown as a cancer of pigment producing cells.

As indicate, supra, tumor rejection antigens, such as the one presentedin SEQ ID NO: 4, are also part of the invention. Also a part of theinvention are polypeptides, such as molecules containing from 8 to 16amino acids, where the polypeptides contain the amino acid sequence setforth in SEQ ID NO: 4. As the examples indicate, those peptides whichare longer than the octamer of SEQ ID NO: 4 are processed into the tumorrejection antigen of SEQ ID NO: 4 by the HLA-Cw6 presenting cancercells, and presented thereby. The presentation leads to lysis bycytolytic T lymphocytes present in a body fluid sample contacted to thecells presenting the complex. Similarly, the peptides longer than SEQ IDNO: 22, such as SEQ ID NO: 21, are processed to the appropriate TRA, andare presented by cancer cells, such as HLA-A29 positive cells.

Thus, another feature of the invention are peptides which are anywherefrom 9 to 16 amino acids long, and comprise the sequence:

Xaa Xaa Trp Pro Xaa Xaa Xaa Xaa Tyr (SEQ ID NO:23) or Xaa Xaa Trp XaaArg Xaa Xaa Xaa Tyr (SEQ ID NO:24) or Xaa Xaa Trp Xaa Xaa Xaa Xaa ArgTyr (SEQ ID NO:25)where Xaa in each case is any amino acid.

Especially preferred are peptides which, in accordance with the formulaof SEQ ID NOS: 23, 24 and 25 also satisfy one or more of the followingcirteria: the N-terminal amino acid position is Tyr, second position isTyr, fourth position is Pro, fifth position is Arg, sixth position isPro, seventh position is Arg, and eighth position is Arg. Of course, thefourth position is already fixed in SEQ ID NO: 23, the fifth is alreadyfixed in SEQ ID No:24, and the eighth in SEQ ID NO: 25. When all ofthese criteria are satisfied and this peptide consists of 9 amino acids,one has SEQ ID NO: 22. Any or al of the foreign specific alternativesmay be combined in the peptides of the claimed invention, subject to themotif of SEQ ID NO: 23, 24 or 25 and the size of 9-16 amino acids.Especially preferred are peptides which are 9-14 amino acids long, andwhich include SEQ ID NO: 23, 24 or 25, subject to the above preferredalternatives.

Also a part of the invention are so-called “minigenes”, which areisolated nucleic acid molecules which encode any of SEQ ID NOS: 21, 22,23, 24 or 25 all of the especially preferred embodiments of SEQ ID NO:23, 24 or 25 being included. There are only a limited number of nucleicacid molecules which can encode, e.g., SEQ ID NO: 21 or 22, and they canall be deduced from the known rules of degeneracy for codons without anydifficulty. The use of these minigenes in encoding the peptides of theinvention, in both standard in vivo and in vitro methodologies is alsoencompassed by the invention. These peptides bind to, and/or areprocessed to peptides which bind to HLA-A29 molecules. The fact thatthese peptides are processed to the tumor rejection antigen in indicatedby the examples.

This property may be exploited in the context of other parameters inconfirming diagnosis of pathological conditions, such as cancer,melanoma in particular. For example, the investigator may study antigensshed into blood or urine, observe physiological changes, and thenconfirm a diagnosis of melanoma using the CTL proliferationmethodologies described herein.

Also a part of the invention are complexes of HLA-A29 molecules, and oneof the peptides listed supra, preferably in soluble form. Such solublecomplexes can be used, e.g., to determine presence of CTLs in a sample,such as a body fluid sample, by adding the soluble complexes to thesample, and then determining reaction with CTLs. Panning for CTLs usingsoluble complexes of MHC molecule and peptide is taught by, e.g., Boussoet al., Immunol. Lett. 59(2): 85-91 (1997), incorporated by reference.The complexes are preferably immobilized to facilitate the enrichment ofthe CTLs. Attention is also drawn to Sakita, et al., J. Immunol. Meth.192: 105-115 (1996), also incorporated by reference, which shows thatsuch complexes can be used to stimulate CTLs in vivo. This is anotherfeature of the invention. In an especially preferred embodiment, thesoluble complexes referred to supra are multimeric, most preferablytetrameric. Altman et al., Science 274: 94-96 (1996), incorporated byreference, describe how such structures can be made.

On their own, peptides in accordance with the invention may be used tocarry out HLA-typing assays. It is well known that when a skin graft,organ transplant, etc., is necessary one must perform HLA typing so asto minimize the possibility of graft rejection. The peptides of theinvention may be used to determine whether or not an individual isHLA-Cw6 or HLA-A29 positive, so that appropriate donors may be selected.This type of assay is simple to carry out. The peptides of the inventionare contacted to a sample of interest, and binding to cells in thatsample indicates whether or not the individual from which the sample istaken is HLA-Cw6 or HLA-A29 positive. One may label the peptidesthemselves, conjugate or otherwise bind them to linkers which arelabeled, immobilize them to solid phases, and so forth, so as tooptimize such an assay. Other standard methodologies will be clear tothe skilled artisan, and need not be presented herein.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-A29 or HLA-Cw6 cells. One such approach isthe administration of CTLs specific to the complex to a subject withabnormal cells of the phenotype at issue. It is within the skill of theartisan to develop such CTLs in vitro. Specifically, a sample of cells,such as blood cells, are contacted to a cell presenting the complex andcapable of provoking a specific CTL to proliferate. The target cell canbe a transfectant, such as COS cell of the type described supra. Thesetransfectants present the desired complex on their surface and, whencombined with a CTL of interest, stimulate its proliferation. COS cells,such as those used herein, are widely available, as are other suitablehost cells.

To detail the therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917 (1986); Riddel et al., Science 257:238 (Jul. 10, 1992); Lynch et al., Eur. J. Immunol. 21: 1403-1410(1991); Kast et al., Cell 59: 603-614 (Nov. 17, 1989)), cells presentingthe desired complex are combined with CTLs leading to proliferation ofthe CTLs specific thereto. The proliferated CTLs are then administeredto a subject with a cellular abnormality which is characterized bycertain of the abnormal cells presenting the particular complex, wherethe complex contains the pertinent HLA molecule. The CTLs then lyse theabnormal cells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/TRA complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing RNA of the pertinent sequences, in thiscase a GAGE sequence. Once cells presenting the relevant complex areidentified via the foregoing screening methodology, they can be combinedwith a sample from a patient, where the sample contains CTLs. If thecomplex presenting cells are lysed by the mixed CTL sample, then it canbe assumed that a GAGE derived, tumor rejection antigen is beingpresented, and the subject is an appropriate candidate for thetherapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach, i.e., the use of non-proliferativecells expressing the complex, has been elaborated upon supra. The cellsused in this approach may be those that normally express the complex,such as irradiated melanoma cells or cells transfected with one or bothof the genes necessary for presentation of the complex. Chen et al.,Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies thisapproach, showing the use of transfected cells expressing HPV E7peptides in a therapeutic regime. Various cell types may be used.Similarly, vectors carrying one or both of the genes of interest may beused. Viral or bacterial vectors are especially preferred. In thesesystems, the gene of interest is carried by, e.g., a Vaccina virus orthe bacteria BCG, and the materials de facto “infect” host cells. Thecells which result present the complex of interest, and are recognizedby autologous CTLs, which then proliferate. A similar effect can beachieved by combining the tumor rejection antigen or the precursoritself with an adjuvant to facilitate incorporation into HLAL-Cw6presenting cells which then present the HLA/peptide complex of interest.The TRAP is processed to yield the peptide partner of the HLA moleculewhile the TRA is presented without the need for further processing.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

1. An isolated nucleic acid molecule which encodes a GAGE tumorrejection antigen precursor, the nucleotide sequence of which is setforth in SEQ ID NO: 14, 15, 16, 17, or
 18. 2. An expression vectoruseful in producing a GAGE tumor rejection antigen, comprising theisolated nucleic acid molecule of claim 1, operably linked to apromoter.
 3. An isolated cell, transformed or transfected with theisolated nucleic acid molecule of claim
 1. 4. An isolated cell,transformed or transfected with the expression vector of claim
 2. 5. Theexpression vector of claim 2, further comprising a nucleic acid moleculewhich encodes an HLA molecule.
 6. The expression vector of claim 5,wherein said HLA molecule is HLA-Cw6 or HLA-A29.
 7. A kit useful intransfecting or transforming a cell comprising: (i) the isolated nucleicacid molecule of claim 1, and (ii) an isolated nucleic acid moleculewhich encodes an HLA molecule.
 8. The kit of claim 7, wherein said HLAmolecule is HLA-Cw6 or HLA-A29.
 9. The isolated nucleic acid molecule ofclaim 1, the nucleotide sequence of which is set forth in SEQ ID NO: 14.